This invention relates to compositions and methods for the inhibition of expression of the ras gene, a naturally occurring gene which occasionally converts to an activated form that has been implicated in tumor formation. This invention is also directed to the specific inhibition of expression of the activated form of the ras gene. This invention is further directed to the detection of both normal and activated forms of the ras gene in cells and tissues, and can form the basis for research reagents and kits both for research and diagnosis. Furthermore, this invention is directed to treatment of such conditions as arise from activation of the ras gene. This invention also relates to stabilized oligonucleotides for inhibition of expression of the ras gene, to such oligonucleotides which have been further modified to enhance their affinity for the ras RNA target, and to such oligonucleotides which have been still further modified to yield sequence-specific elimination of the ras RNA target.
Alterations in the cellular genes which directly or indirectly control cell growth and differentiation are considered to be the main cause of cancer. There are some thirty families of genes, called oncogenes, which are implicated in human tumor formation. Members of one such family, the ras gene family, are frequently found to be mutated in human tumors. In their normal state, proteins produced by the ras genes are thought to be involved in normal cell growth and maturation. Mutation of the ras gene, causing an amino acid alteration at one of three critical positions in the protein product, results in conversion to a form which is implicated in tumor formation. A gene having such a mutation is said to be xe2x80x9cactivated.xe2x80x9d It is thought that such a point mutation leading to ras activation can be induced by carcinogens or other environmental factors. Over 90% of pancreatic adenocarcinomas, about 50% of adenomas and adenocarcinomas of the colon, about 50% of adenocarcinomas of the lung and carcinomas of the thyroid, and a large fraction of malignancies of the blood such as acute myeloid leukemia and myelodysplastic syndrome have been found to contain activated ras oncogenes. Overall, some 10 to 20% of human tumors have a mutation in one of the three ras genes (H-ras, K-ras, or N-ras).
It is presently believed that inhibiting expression of activated oncogenes in a particular tumor cell might force the cell back into a more normal growth habit. For example, Feramisco et al., Nature 1985, 314, 639-642, demonstrated that if cells transformed to a malignant state with an activated ras gene are microinjected with antibody which binds to the protein product of the ras gene, the cells slow their rate of proliferation and adopt a more normal appearance. This has been interpreted as support for the involvement of the product of the activated ras gene in the uncontrolled growth typical of cancer cells.
The H-ras gene has recently been implicated in a serious cardiac arrhythmia called long Q-T syndrome, a hereditary condition which often causes sudden death if treatment is not given immediately. Frequently there are no symptoms prior to the onset of the erratic heartbeat. Whether the H-ras gene is precisely responsible for long Q-T syndrome is unclear. However, there is an extremely high correlation between inheritance of this syndrome and the presence of a particular variant of the chromosome 11 region surrounding the H-ras gene. Therefore, the H-ras gene is a useful indicator of increased risk of sudden cardiac death due to the long Q-T syndrome.
There is a great desire to provide compositions of matter which can modulate the expression of the ras gene, and particularly to provide compositions of matter which specifically modulate the expression of the activated form of the ras gene. It is greatly desired to provide methods of diagnosis and detection of the ras gene in animals. It is also desired to provide methods of diagnosis and treatment of conditions arising from ras gene activation. In addition, improved research kits and reagents for detection and study of the ras gene are desired.
Inhibition of oncogene expression has been accomplished using retroviral vectors or plasmid vectors which express a 2-kilobase segment of the Ki-ras protooncogene RNA in antisense orientation. Mukhopadhyay, T. et al. (1991) Cancer Research 51, 1744-1748; PCT Patent Application PCT/US92/01852 (WO 92/15680); Georges, R. N. et al. (1993) Cancer Research, 53, 1743-1746.
Antisense oligonucleotide inhibition of oncogenes has proven to be a useful tool in understanding the roles of various oncogene families. Antisense oligonucleotides are small oligonucleotides which are complementary to the xe2x80x9csensexe2x80x9d or coding strand of a given gene, and as a result are also complementary to, and thus able to stably and specifically hybridize with, the mRNA transcript of the gene. Holt et al., Mol. Cell Biol. 1988, 8, 963-973, have shown that antisense oligonucleotides hybridizing specifically with mRNA transcripts of the oncogene c-myc, when added to cultured HL60 leukemic cells, inhibit proliferation and induce differentiation. Anfossi et al., Proc. Natl. Acad. Sci. 1989, 86, 3379-3383, have shown that antisense oligonucleotides specifically hybridizing with mRNA transcripts of the c-myb oncogene inhibit proliferation of human myeloid leukemia cell lines. Wickstrom et al., Proc. Nat. Acad. Sci. 1988, 85, 1028-1032, have shown that expression of the protein product of the c-myc oncogene as well as proliferation of HL60 cultured leukemic cells are inhibited by antisense oligonucleotides hybridizing specifically with c-myc mRNA. U.S. Pat. No.: 4,871,838 (Bos et al.) discloses oligonucleotides complementary to a mutation in codon 13 of N-ras to detect said mutation. U.S. Pat. No.: 4,871,838 (Bos et al.) discloses molecules useful as probes for detecting a mutation in DNA which encodes a ras protein.
In all these cases, instability of unmodified oligonucleotides has been a major problem, as they are subject to degradation by cellular enzymes. PCT/US88/01024 (Zon et al.) discloses phosphorothioate oligonucleotides hybridizable to the translation initiation region of the amplified c-myc oncogene to inhibit HL-60 leukemia cell growth and DNA synthesis in these cells. Tidd et al., Anti-Cancer Drug Design 1988, 3, 117-127, evaluated methylphosphonate antisense oligonucleotides hybridizing specifically to the activated N-ras oncogene and found that while they were resistant to biochemical degradation and were nontoxic in cultured human HT29 cells, they did not inhibit N-ras gene expression and had no effect on these cells. Chang et al. showed that both methylphosphonate and phosphorothioate oligonucleotides hybridizing specifically to mRNA transcripts of the mouse Balb-ras gene could inhibit translation of the protein product of this gene in vitro. Chang et al., Anti-Cancer Drug Design 1989, 4, 221-232; Brown et al., Oncogene Research 1989, 4, 243-252. It was noted that Tm was not well correlated with antisense activity of these oligonucleotides against in vitro translation of the ras p21 protein product. Because the antisense oligonucleotides used by Chang et al. hybridize specifically with the translation initiation region of the ras gene, they are not expected to show any selectivity for activated ras and the binding ability of these oligonucleotides to normal (wild-type) vs. mutated (activated) ras genes was not compared.
Helene and co-workers have demonstrated selective inhibition of activated (codon 12 Gxe2x86x92T transition) H-ras mRNA expression using a 9-mer phosphodiester linked to an acridine intercalating agent and/or a hydrophobic tail. This compound displayed selective targeting of mutant ras message in both Rnase H and cell proliferation assays at low micromolar concentrations. Saison-Behmoaras, T. et al., EMBO J. 1991, 10, 1111-1118. Chang and co-workers disclose selective targeting of mutant H-ras message; this time the target was H-ras codon 61 containing an Axe2x86x92T transversion and the oligonucleotide employed was either an 11-mer methylphosphonate or its psoralen derivative. These compounds, which required concentrations of 7.5-150 xcexcM for activity, were shown by immunoprecipitation to selectively inhibit mutant H-ras p21 expression relative to normal p21. Chang et al., Biochemistry 1991, 30, 8283-8286.
Modified nucleotides which increase xcex94xcex94Gxc2x037 for base mismatches can be used to increase selectivity. It has been found that xcex94xcex94Gxc2x037 ranges from 1-2 kcal/mol for the most stable mismatches to 5-6 kcal/mol for the least stable mismatches. When possible, therefore, to maximize selectivity for the mutant target, mutations that generate stable mismatches (e.g., Gxe2x86x92A) are less preferred than mutations that generate unstable mismatches (e.g., Cxe2x86x92G, Uxe2x86x92G, Axe2x86x92C). An example of this can be found in the autosomal dominant mutations associated with familial Alzheimer""s disease. Three different point mutations of the xcex2-amyloid precursor gene have been shown to cosegregate with this disease. These mutations include Gxe2x86x92A (xcex94xcex94Gxc2x037=+1.2 kcal/mol), Gxe2x86x92T (xcex94xcex94Gxc2x037=+3.9 kcal/mol), and Txe2x86x92G (xcex94xcex94Gxc2x037=+6.3 kcal/mol)2. Goate et al., Nature 1991, 349, 704-706; Murrel et al., Science 1991, 254, 97-99; Chartier-Harlin et al., Nature 1991, 353, 844-846. In this case, targeting the Txe2x86x92G mutation is believed to yield the greatest selectivity for mutant xcex2-amyloid by an antisense oligonucleotide.
DNA oligonucleotides having unmodified phosphodiester internucleoside linkages or modified phosphorothioate internucleoside linkages are substrates for cellular RNase H; i.e., they activate the cleavage of target RNA by the RNase H. (Dagle, J. M, Walder, J. A. and Weeks, D. L., Nucleic Acids Research 1990, 18, 4751; Dagle, J. M., Weeks, D. L. and Walder, J. A., Antisense Research And Development 1991, 1, 11; and Dagle, J. M., Andracki, M. E., DeVine, R. J. and Walder, J. A., Nucleic Acids Research 1991, 19, 1805). RNase H is an endonuclease that cleaves the RNA strand of RNA:DNA duplexes; activation of this enzyme therefore results in cleavage of the RNA target, and thus can greatly enhance the ability of antisense oligonucleotides to inhibit target RNA expression. Walder et al. note that in Xenopus embryos, both phosphodiester linkages and phosphorothioate linkages are also subject to exo-nuclease degradation. Such nuclease degradation is detrimental since it rapidly depletes the oligonucleotide available for RNase H activation. PCT Publication WO 89/05358, Walder et al., discloses DNA oligonucleotides modified at the 3xe2x80x2 terminal internucleoside linkage to make them resistant to nucleases while remaining substrates for RNAse H.
Attempts to take advantage of the beneficial properties of oligonucleotide modifications while maintaining the substrate requirements for RNase H have led to the employment of chimeric oligonucleotides. Giles, R. V. et al., Anti-Cancer Drug Design 1992, 7, 37; Hayase, Y. et al., Biochemistry 1990, 29, 8793; Dagle, J. M. et al., Nucleic Acids Research 1990, 18, 4751; Dagle, J. M. et al., Nucleic Acids Research 1991, 19, 1805. Chimeric oligonucleotides contain two or more chemically distinct regions, each comprising at least one nucleotide. These oligonucleotides typically contain a region of modified nucleotides that confer one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the RNA target) and an unmodified region that retains the ability to direct RNase H cleavage. This approach has been employed for a variety of backbone modifications, most commonly methylphosphonates, which alone are not substrates for RNAse H. Methylphosphonate oligonucleotides containing RNase H-sensitive phosphodiester linkages were found to be able to direct target RNA cleavage by RNase H in vitro. Using E. coli RNase H, the minimum phosphodiester length required to direct efficient RNase H cleavage of target RNA strands has been reported to be either three or four linkages. Quartin, R. S. et al. Nucleic Acids Research 1989, 17, 7253; Furdon, P. J. et al. Nucleic Acids Research 1989, 17, 9193. Similar studies have been reported using in vitro mammalian RNase H cleavage assays. Agrawal, S. et al., Proc. Natl. Acad. Sci. USA 1990, 87, 1401. In this case, a series of backbone modifications, including methylphosphonates, containing different phosphodiester lengths were examined for cleavage efficiency. The minimum phosphodiester length required for efficient RNase H cleavage directed by oligonucleotides of this nature is five linkages. More recently, it has been shown that methylphosphonate/phosphodiester chimeras display increased specificity and efficiency for target RNA cleavage using E. coli RNase H in vitro. Giles, R. V. et al., Anti-Cancer Drug Design 1992, 7, 37. These compounds have also been reported to be effective antisense inhibitors in Xenopus oocytes and in cultured mammalian cells. Dagle, J. M. et al., Nucleic Acids Res. 1990, 18, 4751; Potts, J. D., et al., Proc. Natl. Acad. Sci. USA 1991, 88, 1516.
PCT Publication WO 90/15065, Froehler et al., discloses chimeric oligonucleotides xe2x80x9ccappedxe2x80x9d at the 3xe2x80x2 and/or the 5xe2x80x2 end by phosphoramidite, phosphorothioate or phosphorodithioate linkages in order to provide stability against exonucleases while permitting RNAse H activation. PCT Publication WO 91/12323, Pederson et al., discloses chimeric oligonucleotides in which two regions with modified backbones (methyl phosphonates, phosphoromorpholidates, phosphoropiperazidates or phosphoramidates) which do not activate RNAse H flank a central deoxynucleotide region which does activate RNAse H cleavage. 2xe2x80x2-deoxy oligonucleotides have been stabilized against nuclease degradation while still providing for RNase H activation by positioning a short section of phosphodiester linked nucleotides between sections of backbone-modified oligonucleotides having phosphoramidate, alkylphosphonate or phosphotriester linkages. Dagle, J. M, Walder, J. A. and Weeks, D. L., Nucleic Acids Research 1990, 18, 4751; Dagle, J. M., Weeks, D. L. and Walder, J. A., Antisense Research And Development 1991, 1, 11; and Dagle, J. M., Andracki, M. E., DeVine, R. J. and Walder, J. A., Nucleic Acids Research 1991, 19, 1805. While the phosphoramidate containing oligonucleotides were stabilized against exonucleases, each phosphoramidate linkage resulted in a loss of 1.6xc2x0 C. in the measured Tm value of the phosphoramidate containing oligonucleotides. Dagle, J. M., Andracki, M. E., DeVine, R. J. and Walder, J. A., Nucleic Acids Research 1991, 19, 1805. Such loss of the Tm value is indicative of a decrease in the hybridization between the oligonucleotide and its target strand.
Saison-Behmoaras, T., Tocque, B. Rey, I., Chassignol, M., Thuong, N. T. and Helene, C., EMBO Journal 1991, 10, 1111, observed that even though an oligonucleotide was a substrate for RNase H, cleavage efficiency by RNase H was low because of weak hybridization to the mRNA.
Chimeric oligonucleotides are not limited to backbone modifications, though chimeric oligonucleotides containing 2xe2x80x2 ribose modifications mixed with RNase H-sensitive deoxy residues have not been as well characterized as the backbone chimeras. EP Publication 260,032 (Inoue et al.) and Ohtsuka et al., FEBS Lett. 1987, 215, 327-330, employed 2xe2x80x2-O-methyl oligonucleotides (which alone would not be substrates for RNAse H) containing unmodified deoxy gaps to direct cleavage in vitro by E. coli RNase H to specific sites within the complementary RNA strand. These compounds required a minimum deoxy gap of four bases for efficient target RNA cleavage. However, oligonucleotides of this nature were not examined for cleavage efficiency using mammalian RNase H nor tested for antisense activity in cells. These oligonucleotides were not stabilized against nucleases.
Studies on the ability to direct RNase H cleavage and antisense activity of 2xe2x80x2 ribose modifications other than O-methyl have been extremely limited. Schmidt, S. et al., Biochim. Biophys. Acta 1992, 1130, 41.
While it has been recognized that cleavage of a target RNA strand using an antisense oligonucleotide and RNase H would be useful, nuclease resistance of the oligonucleotide and fidelity of the hybridization are also of great importance. There has been a long-felt need for methods or materials that could both activate RNase H while concurrently maintaining or improving hybridization properties and providing nuclease resistance. There remains a long-felt need for such methods and materials for enhancing antisense activity.
It is an object of the invention to provide oligonucleotides complementary to ras mRNA which inhibit expression of the ras gene.
It is another object of the invention to provide oligonucleotides complementary to ras mRNA which specifically inhibit expression of an activated (mutant) form of the ras gene.
Yet another object of the invention is to provide stabilized oligonucleotides which inhibit expression of the ras gene.
Another object of the invention is to provide stabilized oligonucleotides complementary to ras mRNA and modified to increase their affinity for the ras mRNA target, which inhibit expression of the ras gene.
Still another object is to provide oligonucleotides which are complementary to ras mRNA and which are substrates for RNAse H.
An additional object of the invention is to provide oligonucleotides which inhibit proliferation of cancer cells. Methods of inhibiting proliferation of cancer cells are also an object of this invention.
Detection of the mutation from the normal (wild-type) to activated form of the ras gene is another object of the invention.
Differential diagnosis of morphologically similar tumors and identification of high-risk conditions based on the presence of the activated ras gene is yet another object of this invention.
A further object of this invention is to provide methods of diagnosis and treatment of conditions arising due to mutation of the gene from the wild-type to the mutant, activated form of the ras gene.
In accordance with the present invention oligonucleotides having modified internucleosidic linkages or modified nucleosidic units are provided that are complementary to DNA or RNA. In one preferred embodiment, oligonucleotides that are complementary to DNA or RNA deriving from the human H-ras gene are provided. In certain preferred oligonucleotides of the invention are oligonucleotides having alternating linkages or chimeric oligonucleotides having alternating linkages.
It is preferred that these oligonucleotides be complementary to the translation initiation codon of the gene, and preferably that the oligonucleotides comprise a sequence CAT. In accordance with another preferred embodiment, oligonucleotides that are complementary to codon 12 of the activated H-ras gene are provided, preferably comprising a sequence GAC. In another such embodiment, oligonucleotides are provided that are complementary to and hybridize preferentially with the mutated codon 12 of the activated H-ras gene. In this embodiment, such oligonucleotide preferably comprises a sequence GAC.
Oligonucleotides of the invention are conveniently and desirably presented in a pharmaceutically acceptable carrier.
One preferred group of oligonucleotides of the inventions have at least one methylene(methylimino) linkage.
A further preferred group oligonucleotides of the invention have at least one methylene(methylimino) linkage alternating with a phosphorothioate or phosphodiester linkage. An even further group of oligonucleotides of the invention are chimeric oligonucleotides having from 8 to 30 nucleotide units specifically hybridizable with selected DNA or mRNA and containing a first region having at least one methylene(methylimino) linkage and a second region having at least one phosphorothioate linkage. In a more preferred group of these chimeric oligonucleotides the second region is flanked by two of the first regions each of which includes at least one methylene(methylimino) linkage.
In a further preferred groups of chimeric oligonucleotide of the invention the first region includes at least one nucleosidic unit modified at the 2xe2x80x2 position. Preferred for the 2 modifications are 2xe2x80x2-O-alkyl, 2xe2x80x2-O-substituted alkyl or 2xe2x80x2-fluoro modifications. Most preferred is 2xe2x80x2-O-methoxyethyl modifications. In the chimeric oligonucleotides of the invention, preferred oligonucleotides include the second region comprising 2xe2x80x2-deoxynucleotides. A first preferred length for this second region is at least four nucleotides long. A more preferred length for this second region is from five to nine nucleotides long.
In certain preferred chimeric oligonucleotides of the invention the first region includes at least one methylene(methylimino) linkage alternating with a phosphorothioate or phosphodiester linkage. In further preferred oligonucleotides of the invention the first region includes one, two or three methylene(methylimino) linkages alternating with phosphorothioate or phosphodiester linkages.
In accordance with other preferred embodiments, oligonucleotides complementary to ras mRNA are provided which inhibit ras expression and which, at once, have increased resistance to nucleases, have increased binding affinity for the ras mRNA target, and are substrates for RNAse H.
It is presently preferred that increased binding affinity is conveyed by modification of at least one nucleotide at the 2xe2x80x2 position of the sugar, most preferably comprising a 2xe2x80x2-O-alkyl, 2xe2x80x2-O-alkylamino, 2xe2x80x2-O-alkyl-O-alkyl or 2xe2x80x2-fluoro modification.
In some preferred embodiments, the oligonucleotides of the invention are chimeric oligonucleotides comprising at least one region which is modified to increase binding affinity for the complementary ras mRNA, and a region which is a substrate for RNAse H cleavage. In one such embodiment an RNAse H substrate region is flanked by two regions having increased ras mRNA binding affinity.
Other aspects of the invention are directed to methods for modulating the expression of the human ras gene in cells or tissues and for specifically modulating the expression of the activated ras gene in cells or tissues suspected of harboring a mutation leading to such activation.
Some embodiments of the invention are directed to methods for inhibiting the expression of the ras gene and for specifically inhibiting the expression of the activated ras gene.
Additional aspects of the invention are directed to methods of detection of the ras gene in cells or tissues and specific detection of the activated ras gene in cells or tissues suspected of harboring said mutated gene. Such methods comprise contacting cells or tissues suspected of containing the human ras gene with oligonucleotides in accordance with the invention in order to detect said gene.
Other aspects of the invention are directed to methods for diagnostics and therapeutics of animals suspected of having a mutation leading to activation of the ras gene. Such methods comprise contacting the animal or cells or tissues or a bodily fluid from the animal with oligonucleotides in accordance with the invention in order to inhibit the expression of this gene, to treat conditions arising from activation of this gene, or to effect a diagnosis thereof.